Methods for cleaving a gene at a desired site and those for selectively ligating each gene (or DNA) are indispensable in gene manipulation. A typical example of the former is cleaving by the corresponding restriction enzyme, and further, according to needs, sequential cleaving of mono-nucleotides using exonucleases, provided there is a site that recognizes a restriction enzyme close to the desired site.
On the other hand, examples for the methods for ligating at a specific site are DNA ligase-mediated ligations of a cohesive end of a DNA fragment cleaved by a restriction enzyme, or a blunt-end of a DNA fragment or a blunt-end to which a suitable adapter has been added.
The method of utilizing a cohesive end cannot be applied if a restriction enzyme site corresponding to a target gene is not present, and the method of using a blunt-end requires manipulations such as isolating an objective ligate from other ligates, as the direction of ligation cannot be pinpointed.
The method of using an adapter, can be applied regardless of the type of the DNA nucleotide sequence to be ligated, however, the manipulations are complicated, requiring multistage manipulations like adapter-ligation, removal of unreacted adapters, phosphorylation, and such, and also requires some experience in these manipulations. Particularly, it is increasingly complicated when cloning just the objective gene from PCR products, as treatment of byproducts contaminating the objective PCR product becomes necessary because of the properties of PCR. The blunt-end itself that is obtained by PCR can indeed be made available for the ligation, but in this case, in addition to the need of treatments for the above-mentioned byproducts, other problems also arise, such as the direction of the ligation becoming random.
Without using restriction enzymes and DNA ligase, C. Aslanidis et al. (in Nucleic Acids Research, Vol. 18 (1990), 6069–6073) propose a method that comprises forming a bimolecular association product (circular) by a special method via single-stranded (ss) tails of a PCR product amplified using a unique repeated sequence-comprising primer, and a plasmid vector amplified in the same manner; introducing said association product into a bacteria; and selectively obtaining a circular recombinant. However, since this method requires use of a vector comprising a unique repeated sequence and a PCR primer having a corresponding sequence, and also requires single-stranding the ends of PCR products by enzymes following PCR by a special method, it cannot be said to be a versatile method.